Correlation between metastatic potential and variants from colorectal tumor cell line HT-29

AIM: To evaluate the relationship between uPA, PAI-1,CEA, PI3K and metastatic potential in three colorectal tumor cell lines.METHODS: Metastatic model in nude rats was established by variants HT-29c and HT-29d cell lines and the metastatic potential of two tumor cell variants was compared.Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined using ELISA in colorectal carcinoma WiDr, HT29 and HT-29d cell lines with different metastatic potentials.Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) was analyzed using immunohistochemistry (IHC) in these cell lines in vitro and in vivo. CEA expression was compared using fluorescence activated cell sorter (FACS)in vitro.RESULTS: The number of HT-29d cells arrested in liver dramatically decreased within the initial 24 hours after injection. The taking rate of liver metastases in the variant HT-29d increased as compared with parental HT-29 cells (70 % versus 50 %) and a variant HT-29b cells (70 % versus 60 %), and extensive organs were synchronously involved in metastases. The uPA concentration of variant HT-29d cell line was significantly higher than that of the non-metastatic WiDr and the low metastatic HT-29 cell lines. The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-metastatic WiDr cells and the low metastatic HT-29 cellsin vivo.CONCLUSION: The selected variant HT-29d cell exhibited an enhanced metastatic potential. The level of uPA and PAI-1 is positively correlated with the metastatic capacity of tumor cells. The expression of PI3-kinase correlates with tumor development and metastasis.

Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

绿色荧光蛋白在人大肠癌HT-29c细胞的表达

目的 观察增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)在人大肠癌细胞的表达情况。 方法 构建逆转录病毒载体质粒prkatEGFP/neo ,用磷酸钙沉淀法转导 2 93T细胞 ,4 8h获得高滴度病毒颗粒上清液并转染人大肠癌HT 2 9c细胞。荧光显微镜及流式细胞仪观察转染的EGFP基因在人大肠癌HT 2 9c细胞体内外的表达。 结果 携带EGFP的逆转录病毒载体能有效地转染人大肠癌细胞 ,HT 2 9c细胞能稳定、高效和持久地表达EGFP ,与未转染细胞比较 ,他们的生物学特性未改变 (F =0 6 2 ,P >0 0 5 )。在裸大鼠皮下肿瘤中 ,EGFP也能稳定、高效的表达。 结论 外源性EGFP能在人大肠癌HT 2 9c细胞内进行有效的复制、转录和翻译 ,在体内外可持续、稳定、高效的表达。它能作为一种新的实验方法应用于肿瘤细胞生长。